Discovery of N-(4-(3-amino-1H-indazol-4-yl)phenyl)-N′-(2-fluoro-5- methylphenyl)urea (ABT-869), a 3-aminoindazole-based orally active multitargeted receptor tyrosine kinase inhibitor

Article abstract of DOI:10.1021/jm061280h

In our continued efforts to search for potent and novel receptor tyrosine kinase (RTK) inhibitors as potential anticancer agents, we discovered, through a structure-based design, that 3-aminoindazole could serve as an efficient hinge-binding template for kinase inhibitors. By incorporating an N,N′-diaryl urea moiety at the C4-position of 3-aminodazole, a series of RTK inhibitors were generated, which potently inhibited the tyrosine kinase activity of the vascular endothelial growth factor receptor and the platelet-derived growth factor receptor families. A number of compounds with potent oral activity were identified by utilizing an estradiol-induced mouse uterine edema model and an HT1080 human fibrosarcoma xenograft tumor model. In particular, compound 17p (ABT-869) was found to possess favorable pharmacokinetic profiles across different species and display significant tumor growth inhibition in multiple preclinical animal models.

Full text of DOI:10.1021/jm061280h

1584
J. Med. Chem. 2007, 50, 1584-1597
Discovery of N-(4-(3-Amino-1H-indazol-4-yl)phenyl)-N′-(2-fluoro-5-methylphenyl)urea
(ABT-869), a 3-Aminoindazole-Based Orally Active Multitargeted Receptor Tyrosine Kinase
Inhibitor
Yujia Dai,*,† Kresna Hartandi,† Zhiqin Ji,† Asma A. Ahmed,‡ Daniel H. Albert,† Joy L. Bauch,† Jennifer J. Bouska,†
Peter F. Bousquet,‡ George A. Cunha,‡ Keith B. Glaser,† Christopher M. Harris,‡ Dean Hickman,† Jun Guo,† Junling Li,†
Patrick A. Marcotte,† Kennan C. Marsh,† Maria D. Moskey,‡ Ruth L. Martin,† Amanda M. Olson,† Donald J. Osterling,†
Lori J. Pease,† Niru B. Soni,† Kent D. Stewart,† Vincent S. Stoll,† Paul Tapang,† David R. Reuter,† Steven K. Davidsen,† and
Michael R. Michaelides†
Global Pharmaceutical Research and DeVelopment, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064-6100, and Abbott
Bioresearch Center, 100 Research DriVe, Worcester, Massachusetts 01605-5314
ReceiVed NoVember 2, 2006
In our continued efforts to search for potent and novel receptor tyrosine kinase (RTK) inhibitors as potential
anticancer agents, we discovered, through a structure-based design, that 3-aminoindazole could serve as an
efficient hinge-binding template for kinase inhibitors. By incorporating an N,N′-diaryl urea moiety at the
C4-position of 3-aminodazole, a series of RTK inhibitors were generated, which potently inhibited the tyrosine
kinase activity of the vascular endothelial growth factor receptor and the platelet-derived growth factor
receptor families. A number of compounds with potent oral activity were identified by utilizing an estradiol-
induced mouse uterine edema model and an HT1080 human fibrosarcoma xenograft tumor model. In
particular, compound 17p (ABT-869) was found to possess favorable pharmacokinetic profiles across different
species and display significant tumor growth inhibition in multiple preclinical animal models.
Introduction
therapy has promoted even greater interest in this field. Several
KDR-selective small molecule inhibitors, including 1 (PTK787)12
(Figure 1), are in late-stage clinical trials.
Angiogenesis, a process in which new blood vessels are
formed from pre-existing vasculatures, is required for tumor
growth and metastasis because blood is essential for solid tumors
to manage nutritional supplies and waste removal. It has been
shown that angiogenesis is a rate-limiting step in tumor
development. Tumors that lack an adequate vasculature become
necrotic or apoptotic and do not grow beyond a limited size.
Consequently, inhibition of tumor angiogenesis has become a
compelling approach in the development of anticancer agents.1-3
In tumor angiogenesis, vascular endothelial growth factor
receptor tyrosine kinases are thought to play a prominent role
by interacting with their extracellular ligands, vascular endot-
helial growth factors (VEGFs).4 As a principal subfamily of
receptor tyrosine kinases, VEGFRs (vascular endothelial growth
factor receptors) are predominantly expressed in vascular
endothelial cells and consist of FLT1 (Fms-like tyrosine kinase
1; VEGFR1), KDR (VEGFR2), and FLT4 (VEGFR3).5,6 Over-
activation of VEGFR family RTKs and, in particular, of KDR
by VEGFs has been linked to the progression of a variety of
human cancers. Studies have shown that VEGF-mediated KDR
signaling induces a series of endothelial responses such as
proliferation, migration, and survival and ultimately leads to
new vessel formation and maturation. Due to the vital role that
KDR signaling plays in tumor angiogenesis, compounds that
have the ability to interrupt KDR signaling by targeting KDR
or its ligands (VEGFs) have been pursued for the development
of antiangiogenesis-based agents.7-11 The approval by the FDA
of Bevacizumab, a VEGF antibody, for the treatment of first-
line metastatic colorectal cancer in combination with chemo-
Although VEGFRs play an important role in tumor growth
and metastasis by means of angiogenesis, overactivation of other
RTKs also contributes to tumor progression through a variety
of mechanisms. One subfamily of these RTKs is the platelet-
derived growth factor receptor tyrosine kinases, which are
structurally related to the VEGFR family and include PDGFRR,
PDGFRâ, cKIT, CSF1R (colony-stimulating factor 1 receptor),
and FLT3. PDGFR (platelet-derived growth factor receptor)
kinases are believed to not only indirectly promote tumor
angiogenesis, but also directly contribute to tumor growth by
modifying the tumor microenvironment.13-15 Additionally, the
constitutive activation of FLT3 and cKIT by mutation is directly
associated with the progression of acute myeloid leukemia
(AML)16 and gastrointestinal stromal tumor (GIST).17
As a result of the complex and redundant cellular signaling
network associated with RTKs, broad-acting, multitargeted RTK
inhibitors may be even more advantageous than selective agents,
because these types of inhibitors have the ability to block
multiple signaling pathways, upon which tumor survival de-
pends.18,19 This is evidenced by the FDA’s recent approval of
two multitargeted kinase inhibitors, 2 (SU11248)20 and 3 (BAY
43-9006)21 (Figure 1), as new agents for cancer treatment. While
2 targets both VEGFR and PDGFR tyrosine kinases, 3 also
inhibits Raf kinase in addition to the VEGFR and PDGFR
kinases.
In the search for novel and potent multitargeted RTK
inhibitors, we recently identified a series of thienopyrimidine
ureas (e.g., 5 in Figure 2) as potent inhibitors of all the kinases
of both VEGFR and PDGFR families.22 A number of com-
pounds from this series were found to be orally efficacious in
tumor growth models such as human HT1080 fibrosarcoma
xenografts. The preliminary but promising results of this series
* To whom correspondence should be addressed. Dr. Yujia Dai, R47J/
AP10, Cancer Research, 100 Abbott Park Road, Abbott Park, Illinois 60064.
Tel.: (847) 937-8977. Fax: (847) 935-5165. E-mail: yujia.dai@abbott.com.
† Abbott Laboratories, Abbott Park, IL.
‡ Abbott Bioresearch Center, Worcester, MA.
10.1021/jm061280h CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/08/2007

NoVel Tyrosine Kinase ActiVity Inhibitor ABT-869
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1585
Figure 1. KDR-selective inhibitor 1 and multitargeted RTK inhibitors 2 and 3.
nucleus in 5 mimicked the adenine component of ATP, forming
a pair of hydrogen-bonding interactions with the KDR hinge
region. The N,N′-diaryl urea portion extended into the hydro-
phobic back pocket of KDR kinase and contributed significantly
to the inhibitors’ KDR affinity. Considering the importance of
the urea link for the potency of thienopyrimidine inhibitors, we
decided to maintain the N,N′-diaryl urea portion in our new
inhibitors and try to replace the thienopyrimidine pharmacophore
with an alternative template. Such a template should possess
two basic structural features: the capability to form hydrogen-
bonding interactions with the KDR hinge region and an
appropriate attachment vector for the diaryl urea, allowing for
optimal interactions with the KDR hydrophobic pocket. With
this working plan in mind, we envisioned that removal of the
CH unit from the six-membered pyrimidine ring (A-ring) and
insertion back into the five-membered B-ring, as shown in
Figure 2, might satisfy both design criteria. This “CH-shift”
strategy converted the 6-5 ring system of the thienopyrimidine
into a 5-6 system, one permutation of which is the 3-aminoin-
dazole (6). With its 3-amino group and the ring nitrogen (N2),
the 3-aminoindazole unit should be able to mimic the adenine
component of ATP to form a pair of hydrogen-bonding
interactions with the KDR hinge region. The endocyclic NH
unit might offer another site for a third hydrogen-bonding
interaction with the kinase. Additionally, incorporation of the
N,N′-diaryl urea at the C4-position of the indazole system should
provide the required orientation for the urea to interact with
the KDR back pocket.
Figure 2. Rational design of 3-aminoindazole urea RTK inhibitors.
To provide support to this rational design, a homology model
of KDR bound to 5 (Figure 3) was created, following the same
protocol as reported previously for the thienopyrimidine urea
KDR inhibitors.22 This model suggested that this new series of
compounds represented by the general structure 6 would, as
expected, bind to the ATP site of an inactive confirmation of
KDR kinase (DFG-out confirmation). The 3-aminoindazole
template indeed mimicked the adenine portion of ATP, interact-
ing with the hinge region of KDR through hydrogen bonds
between (1) the exocyclic amino (3-amino) group and the
backbone carbonyl of Glu 917 and (2) the proximal ring nitrogen
(N2) and the backbone NH of Cys 919. The urea portion
accessed the back hydrophobic pocket adjacent to the ATP-
binding site, with the urea carbonyl oxygen forming a hydrogen-
bonding interaction with the backbone N-H of Asp 1046 of
the DFG motif. The external NH of the urea link also formed
a hydrogen-bonding interaction with the side chain carboxylate
of Glu 885 of the RC helix. These suggested interactions
between the urea unit and the back hydrophobic pocket of KDR
were consistent with reports of other RTK inhibitors of the urea
class23,24 and matched well with the proposed binding model
for the thienopyrimidine ureas. In fact, the modeling was able
to generate an almost perfect overlap for 17b and thienopyri-
midine urea 5 (Figure 4).
Figure 3. Model of 17b bound to KDR kinase. Hydrogen bonds in
black are shown between the urea external NH and Glu 885 carboxylate,
between the 3-amino group of the indazole and Glu 917 backbone
carbonyl, and between the ring nitrogen (N2) and Cys 919 NH. Also
in thick bond are residues Asp 1046-Phe 1047 of the DFG motif in
the inactive conformation (DFG-out).
inspired us to search for additional inhibitors with new chemo-
types to target VEGFR and PDGFR kinases. Utilizing a rational
design strategy, we were able to discover a new series of urea
RTK inhibitors based on a 3-aminoindazole template (6, Figure
2). These 3-aminoindazole ureas proved to be potent inhibitors
against the members of the VEGFR and PDGFR families. From
this series, a clinical candidate, 17p (ABT-869), was identified.
In this paper, we report the rational design, chemical synthesis,
and structure-activity relationships of this novel series as well
as in vitro and in vivo properties of 17p.
Inhibitor Design
In our studies of the thienopyrimidine series, a homology
model of KDR bound to 5 suggested that the thienopyrimidine

1586 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Dai et al.
protected in the form of a phthalimide (Scheme 2). While ureas
22a,c,d were conveniently synthesized via reaction of anilines
21a,c,d with the corresponding aryl isocyanates, the Suzuki
coupling reaction between iodide 20b and urea boronate 16b
was chosen for the preparation of urea 22b to avoid the potential
side reaction of the alcohol functionality in aniline 21b (not
shown in Scheme 2) with the isocyanate.
Analogs with a substituent at the 7-position of the aminoin-
dazole were synthesized as outlined in Scheme 3. Fluoride-
directed lithiation of 23a-d with lithium diisopropylamide
(LDA) and subsequent reaction with carbon dioxide yielded
acids 24a-d, which were then converted into amides 25a-d
via the corresponding acid chlorides. Dehydration of 25a-d to
form nitriles 26a-d was carried out using thionyl chloride in
warm dimethylformide (DMF). With nitriles 26a-d in hand,
anilines 28a-d were readily synthesized via the cyclization with
hydrazine followed by the Pd-mediated coupling. Unlike the
case of aniline 9, the 7-substituents on the indazole ring in
28a-d sterically suppressed the competitive N-acylation reaction
at the N1-position and allowed anilines 28a-d to react with
m-tolyl isocyanate to generate desired ureas 29a-d.
Figure 4. Overlap of 3-aminoindazole urea 17b (in green) with
thieopyrimidine urea 5 (in purple).
Scheme 1. Synthesis of 3-Aminoindazole Amide 13,
Sulfonamide 14, and Ureas 17a-sa
Nitriles 26a and 26b not only served as the intermediates
for the synthesis of ureas 29a and 29b, but also provided access
to analogs with a variety of other substituents at the 7-position
of 3-aminoindazole. Thus, demethylation of 26b with BBr3
followed by an O-alkylation of the produced phenol (30) led to
various O-alkylated products (31a-f), from which ureas 34a-f
were readily synthesized (Scheme 4). Similarly, analogs with
an aminomethyl-linked substituent at the 7-position of 3-ami-
noindazole such as 43a and 43b could be synthesized from
bromide 39, which was prepared via benzylic bromination of
26a (Scheme 5).
Results and Discussion
Kinase enzymatic assays were performed utilizing the ho-
mogeneous time-resolved fluorescence (HTRF) protocol in the
presence of a high concentration (1.0 mM) of ATP. Given that
KDR plays a primary role in tumor angiogenesis, optimization
of potency against KDR was emphasized in the SAR studies.
The first compound of this series tested in the KDR assay was
3-aminoindazole aniline 9. A moderate inhibitory activity (KDR
IC50 ) 4790 nM) was measured for 9 (Table 1). This activity
was very similar to that shown by the corresponding thienopy-
rimidine aniline 4 (KDR IC50 ) 4600 nM), indicating that the
3-aminoindazole nucleus indeed held the potential as a promis-
ing new template for RTK inhibitors. In fact, just as was seen
in the thienopyrimidine series, the KDR potency improved
remarkably when 9 was converted to an N,N′-diaryl urea. Phenyl
urea 17a (KDR IC50 ) 64 nM) was about 75-fold more potent
than 9. A further improved potency was observed for m-tolyl
urea 17b (KDR IC50 ) 3 nM). The substantially improved KDR
potency shown by 17a and 17b was consistent with the proposed
KDR binding model and clearly revealed the importance of the
urea moiety for the KDR affinity. The importance of the urea
link was further demonstrated by the significant loss of potency
suffered by the amide analog (13) and the sulfonamide analog
(14). Compound 14 was almost 88-fold less active against KDR
than 17a, while no apparent inhibitory activity was seen for
13, even at a concentration of 12.5 µM. Additionally, N,N′-
diaryl ureas seemed to be optimal for KDR inhibition. Just as
the phenyl analog (17a) and the m-tolyl analog (17b), 3-thiophene
analog 17c was also a very potent KDR inhibitor. Replacement
of the urea terminal aromatic groups with a cyclopentyl or a
cyclohexyl group still generated fairly potent KDR inhibitors
a Reagents and conditions: (i) NH2NH2‚H2O, n-BuOH, 110 °C, 90%;
(ii) Pd(PPh3)4, Na2CO3, toluene, EtOH, H2O, 90 °C, 60%; (iii) PhXCl, Et3N,
CH2Cl2, 0 °C f rt, 80-95%; (iv) RNCO, CH2Cl2, 0 °C f rt, 70-95%;
(v) RNH2, CH2Cl2, 0 °C f rt; (vi) Method A: Pd(dppf)Cl2‚CH2Cl2, Na2CO3,
DME, H2O, 85 °C; or method B: Pd(dppf)Cl2‚CH2Cl2, Na2CO3, DME,
H2O, microwave, 140-160 °C, 15-20 min.
Chemistry
A general synthesis of C4-substituted aminoindazoles is
shown in Scheme 1. 3-Amino-4-iodoindazole (8) was easily
prepared by reaction of 2-fluoro-6-iodobenzonitrile (7) with
hydrazine monohydrate. The Suzuki coupling reaction between
8 and boronate 10 provided aniline 9. Despite its easy access,
9 could not be used for the synthesis of ureas 17a-s due to a
competitive endocyclic N-acylation at NH-position (N1) of the
indazole upon reaction with isocyanates. Consequently, ureas
17a-s were synthesized by coupling 8 with urea boronates
16a-s, which were prepared either via reaction of 10 with the
corresponding isocyanates or via reaction of 15 with the
corresponding amines. In a similar fashion, amide 13 and
sulfonamide 14 were obtained from reaction of 8 with amide
boronate 11 and sulfonamide boronate 12, respectively.
Introduction of a substituent on the endocyclic NH (N1) of
3-aminoindazoles (20a-d) was accomplished by treating 7 with
an alkyl hydrazine RNHNH2 (R ) Me, HOCH2CH2) or via
alkylation of 18, in which the 3-amino group was selectively

NoVel Tyrosine Kinase ActiVity Inhibitor ABT-869
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1587
Scheme 2. Synthesis of N1-Substituted 3-Aminoindazole Ureas 22a-da
a Reagents and conditions: (i) RNHNH2, n-BuOH, 110 °C, 80-90%; (ii) phthalic anhydride, dioxane, 110 °C, 31%; (iii) RBr, Na2CO3, DMF, 30-60%;
(iv) NH2NH2‚H2O, EtOH, 0 °C f rt, 85-90%; (v) 10, Pd(PPh3)4, Na2CO3, DME, H2O, 90 °C, 45-70%; (vi) aryl isocyanate, DMF, 0 °C f rt, 30-70%;
(vii) 16b, Pd(PPh3)4, Na2CO3, DME, H2O, 90 °C, 39%.
Scheme 3. Synthesis of 7-Substituted 3-Aminoindazole Ureas
29a-da
Scheme 4. Synthesis of 7-Substituted 3-Aminoindazole Ureas
34a-fa
a Reagents and conditions: (i) LDA, THF, -78 °C, 1 h; then solid CO2,
-78 °C f rt, 60-80%; (ii) SOCl2, reflux, 2 h or (COCl)2, cat. DMF, rt;
then concentrated aqueous NH4OH, THF, 0 °C f rt, 90-98%; (iii) SOCl2,
DMF, 115 °C, 60-80%; (iv) NH2NH2‚H2O, n-BuOH, 110 °C, 80-90%;
(v) 10, Pd(PPh3)4, Na2CO3, toluene, EtOH, H2O, 90 °C, or microwave, 140-
160 °C, 20 min, 45-65%; (vi) m-tolyl isocyanate, CH2Cl2, 0 °C f rt, 35-
70%.
a Reagents and conditions: (i) BBr3, CH2Cl2, -78 °C, 77%; (ii) RBr,
K2CO3; or ROH, DEAD, PPh3, THF, 70-97%; (iii) NH2NH2‚H2O, n-BuOH,
110 °C, 50-70%; (iv) 10, Pd(PPh3)4, DME, H2O, 85 °C, or microwave,
140-160 °C, 15-20 min; (v) m-tolyl isocyanate, DMF, 0 °C f rt, 35-
70%.
(17d: IC50 ) 153 nM and 17e: IC50 ) 86 nM), but these
aliphatic ureas had no obvious advantage over their aromatic
analogs.
Further screening of these 3-aminoindazole ureas against other
RTKs showed that they were also potent against other VEGFR
kinases as well as the kinases of the PDGFR family. As the
data in Table 1 show, these ureas were potent inhibitors of both
FLT3 and cKIT. The SAR regarding cKIT was very similar to
that displayed in KDR inhibition; an N,N′-diaryl urea moiety
was also optimal for cKIT affinity. However, the binding affinity
for FLT3 appeared to be less sensitive to the nature of the urea
terminal group. Potent activity against FLT3 was observed for
both aromatic and aliphatic ureas. Given that mutational
activation of FLT3 and cKIT are respectively implicated in AML
and GIST, these compounds have potential as targeted thera-
peutics for these diseases.

1588 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Dai et al.
Table 1. RTK Inhibitory Activity of C4-Substituted Aminoindazoles
Scheme 5. Synthesis of 7-Substituted 3-Aminoindazole Ureas
43a,ba
a Reagents and conditions: (i) NBS, (PhCO2)2, benzene, reflux, 46%;
(ii) amine, DMF, rt, 90-98%; (iii) NH2NH2‚H2O, n-BuOH, 110 °C, 50-
75%; (iv) 10, Pd(PPh3)4, DME, H2O, 85 °C, or microwave, 140-160 °C,
15-20 min; (v) m-tolyl isocyanate, DMF, 0 °C f rt, 40-60%.
Concluding that an N,N′-diaryl urea moiety at the C4-position
of the aminoindazole nucleus was optimal for KDR inhibition,
we then examined the substitution on the urea terminal phenyl
group of 17a. As the results in Table 2 show, incorporation of
a fluoro or a methyl group on the phenyl group was tolerated
at all three different positions (meta-, ortho-, and para-positions);
however, consistent with the thienopyrimidine ureas,22 the meta-
substituted analogs (17b and 17g) were the most potent in terms
of KDR inhibitory activity. In fact, with an IC50 value of 3 nM,
m-tolyl urea 17b was one of the most potent KDR inhibitors of
this series based on the enzymatic assay. Very potent KDR
inhibitory activity was also achieved by introduction of an m-Et
(17k: KDR IC50 ) 6 nM), an m-Cl (17l: KDR IC50 ) 8 nM),
or an m-CF3 (17n: KDR IC50 ) 10 nM). The potency gain
observed for these compounds bearing a meta-substituent in
comparison to 17a seems to be consistent with the modeling
suggestion that an additional hydrophobic volume exists near
the meta-position. Substitution with an m-Br or m-OH is also
tolerated. Introduction of an additional fluoro group on the
m-tolyl ring of 17b and the p-tolyl ring of 17j had no impact
on the KDR affinity, but as discussed later, these analogs (17p,
17q, and 17r) exhibited improved in vivo properties (see the
data in Table 5 and the related discussion). Incorporation of an
additional fluoro group at the meta-position to the CF3 group
on the phenyl in 17n (KDR IC50 ) 10 nM), on the other hand,
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate.
KDR inhibitor, exhibiting an IC50 value of 11 nM (Table 3).
However, significant loss of KDR potency was observed for
all other groups examined (22b-d). The unfavorable steric
interaction imposed by these larger groups might be responsible
for the reduction in potency.
Based on the proposed binding model, a substituent at the
7-position of the 3-aminoindazole nucleus should project toward
solvent and, thus, chemical elaborations aimed at modulating
physiochemical properties at this position should be tolerated.
Indeed, the SAR of this position was consistent with this
prediction (Table 4). Substituents such as Me, MeO, F, and Br
(29a-d) as well as various polar groups that were attached to
the position via a three-atom ether link (CH2CH2O; 34a-f) were
well tolerated. Nonetheless, when the link was shortened to a
methylene unit, reduced RTK inhibitory activity was observed
(43a: KDR IC50 ) 390 nM; 43b: KDR IC50 ) 1200 nM).
Compounds 43a and 43b were not only poor KDR inhibitors,
but also weak against FLT3 and cKIT. In these cases, the
4-methylpiperazino and morpholino groups in close proximity
to the 3-aminoindazole nucleus might disrupt the interaction
between the 3-aminoindazole core and the hinge region of the
kinases.
caused a 9-fold reduction in KDR affinity (17s: KDR IC50
)
90 nM).
The SAR at the endocyclic indazole NH was also explored.
The modeling suggested that there was no direct hydrogen-
bonding interaction between the NH and the KDR protein and,
consequently, elaboration at the NH position might be tolerated.
Indeed, N-methylation of the NH in 17b only caused a slight
drop in KDR potency. Compound 22a was still a very potent
The proposed hydrogen-bonding interactions between the
3-aminoindazole moiety and the two amino acid residues (Glu
917 and Cys 919) of the KDR hinge region should be vital to
the binding affinity of these inhibitors. Interrupting these
interactions would predictably have a large negative impact on
potency. Indeed, this was clearly demonstrated by the significant

NoVel Tyrosine Kinase ActiVity Inhibitor ABT-869
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1589
Table 4. Substitutions at the 7-Position of 3-Aminoindazoles
Table 2. Substitutions at the Terminal Phenyl of the N,N′-Diaryl Ureas
KDR
FLT3
cKIT
cmpd
X
IC50a (nM)
IC50a (nM)
IC50a (nM)
17a
17f
17g
17h
17i
17b
17j
17k
17l
17m
17n
17o
17p
17q
17r
17s
H
64
82
23
67
87
3
12
6
8
36
10
55
4
4
36
90
54
157
25
26
3
44
34
15
20
86
20
17
42
13
69
41
48
16
10
42
150
o-F
m-F
p-F
o-Me
m-Me
p-Me
m-Et
m-Cl
m-Br
m-CF3
m-OH
2-F-5-Me
4-F-3-Me
3-F-4-Me
2-F-5-CF3
4
2
55
3
106
10
26
5
7
47
270
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate.
Table 3. N1-Substitution of 3-Aminoindazoles
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate.
potent than its 3-amino analog 22a. Interestingly, an even more
potent activity was recorded for indazole urea 47 (KDR IC50
)
18 nM), which was only 6-fold less potent than 17b. At this
point, it is not clear whether the hydrogen-bonding interaction
between the indazole ring nitrogen and Cys 919 of KDR became
more optimal in the absence of substituents at both indazole
N1- and C3-positions in 47 or whether the indazole ring NH
also contributes to the potency.
Based on the results of the KDR enzymatic assay, selected
3-aminoindazole ureas were further characterized as indicated
in Table 5. The potent inhibition of these inhibitors against KDR
was generally well reflected at the cellular level. As the results
in Table 5 show, these compounds potently inhibited VEGF-
induced KDR phosphorylation in 3T3-murine fibroblast cells
engineered to express human KDR, mostly exhibiting IC50
values in the low double-digit nanomolar range. It is noteworthy
that the incorporation of various water-solubilizing groups at
the 7-position of the indazole yielded very comparable cellular
KDR activity relative to the KDR enzymatic potency (lower
portion of Table 5).
To evaluate the in vivo activity, compounds with potent
cellular activity were first screened in an estradiol-induced
mouse uterine edema (UE) model.22 This functional assay served
as a useful tool for a rapid and preliminary evaluation of KDR
inhibitors’ oral activity. Among the three methyl-substituted
3-aminoindazole ureas (17b, 17i, and 17j), only the meta-methyl
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate.
loss in KDR potency observed for both 44 and 45 (>1000-fold
and 290-fold, respectively; Figure 5) in comparison to 22a (KDR
IC50 ) 11 nM). It appeared that the added groups at the 3-amino
residue in 44 and 45 not only affected the hydrogen bonding
interaction between the amino group and the Glu 917 of KDR,
but also the one between the indazole ring nitrogen (N2) and
Cys 919 residue, because indazole urea 46, which does not
possess a 3-amino group, was still fairly potent against KDR.
With an IC50 value of 465 nM, 46 was only about 42-fold less

1590 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Dai et al.
Table 5. In Vitro and In Vivo Properties of Selected 3-Aminoindazole Ureas
a Each IC50 determination was performed with seven concentrations, and each assay point was determined in duplicate. b Activity against VEGF-induced
KDR phosphorylation in 3T3-murine fibroblast cells. Each cellular IC50 determination was performed with five concentrations, and each assay point was
determined in duplicate. c Dosed orally in balb/c female mice. d Percent inhibition in mg/kg. e Dosed orally twice a day in SCID-beige mice. f Tumor percent
inhibition as the total daily dose (mg/kg). g Dosed orally at 10 mg/kg in CD-1 mice.
displayed a very similar KDR cellular activity to that shown
by 17b, it produced no inhibition of the edema at the same dose.
It appeared that substitution with a small lipophilic group on
the meta-position of the urea terminal phenyl group not only
gave the best KDR enzymatic potency (cf. data in Table 2),
but also delivered potent in vivo activity. This was further
demonstrated by the efficacy of 17l, 17n, and 22a in the UE
model, with each exhibiting an ED50 value equal to or less than
4 mg/kg. Possessing a 2-fluoro-5-methylphenyl group, com-
pound 17p was extremely potent (ED50 ) 0.5 mg/kg) and
compared favorably in this model to both 5 and 2, which
displayed an ED50 value of 5 mg/kg and 11 mg/kg, respectively.
As was mentioned before, the fluoro group in 17p had no impact
on the KDR enzymatic potency in comparison to 17b; however,
17p showed a significantly enhanced UE potency, which is
consistent with its much improved mouse oral plasma exposure
Figure 5. Indazole ureas 44-47 and their activity against KDR.
(AUC at an oral dose of 10 mg/kg: 24.5 µΜ‚h for 17p vs 6.5
µM‚h for 17b). Incorporation of a fluoro group on the urea
terminal aryl group also led to an improved mouse oral
analog 17b showed potent oral activity in the UE model at an
oral dose of 10 mg/kg. Although the para-methyl analog 17j
pharmacokinetic (PK) profile for 17q and an enhanced UE

NoVel Tyrosine Kinase ActiVity Inhibitor ABT-869
Table 6. Kinase Inhibition Profile of 17p
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1591
8.2 mL/min‚kg) following intravenous (IV) dosing. Its plasma
IV half-lives ranged from 2.0 h in dog to 3.6 h in rat, while
oral bioavailability ranged from 10 to 47%.
kinase
KDR
FLT1
FLT4
PDGFRâ
CSF1R
KIT
IC50a (nM)
kinase
IC50a (nM)
4
3
190
66
3
14
4
170
RET
FGFR
SRC
IGFR
LCK
1,900
12,500
50,000
50,000
50,000
50,000
50,000
9,800
Consistent with its lipophilic character, 17p displayed ex-
tensive plasma protein binding with 96.8% in monkey, 98.2%
in mouse, and 99.1% in rat. In human plasma, the value is
99.0%. Compound 17p did not show significant activity against
the Ikr potassium channel hERG (human ether-a-go-go-related
gene), as measured in a patch clamp assay. Only 5.9% blockage
of hERG current in transfected HEK 293 cells was observed at
a concentration of 0.35 µM. Additionally, no effect on the action
potential duration of canine Purkinje fibers was measured with
17p at a concentration of 5.45 µM using standard in vitro
microelectrode techniques.
FGR
FLT3
TIE2
AKTb
CDC2b
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate. b IC50 values were determined
at an ATP concentration of 5-10 µmol/L.
Table 7. PK Profile of 17p
IV
PO
In vitro metabolism studies of 17p showed that its metabolic
turnover was low in mouse, dog, monkey, and human hepato-
cytes. About 91-92% of the parent drug remained after 6 h
incubation. A number of major metabolites in human hepato-
cytes were identified, which include oxidation products 48 and
49, N-glucoronidation product 50, and oxidation/glucoronidation
product 51 (Figure 6). While the exact hydroxylation positions
in 48 and 51 are not clear yet, the structures of 49 and 50 have
been confirmed. Compound 49 was tested in the KDR enzymatic
assay and showed no significant inhibition of KDR (IC50 ) 6580
nM).
t1/2
(h)
Vd
(L/kg)
Cl
AUC
(µg‚h/mL)
F
(%)
species
(mL/min‚kg)
rata
3.6
3.1
2.0
1.1
2.2
1.2
3.3
8.2
7.2
6.8
0.6
1.5
27.0
10.6
47.0
monkeyb
dogb
a Dosed at 5 mg/kg both intravenously and orally. b Dosed at 2.5 mg/kg
both intravenously and orally.
activity for 17r when compared to 17b and 17j, respectively.
Compounds with a water-solubilizing group at the indazole C7-
position generally appeared less potent in the edema model than
their unsubstituted analogs, in spite of their comparable KDR
cellular activity. The limited mouse oral exposure of 34d and
38 provided one explanation for their poor activity in the edema
model.
With its potent activity against VEGF and PDGF receptor
tyrosine kinases, good oral PK profile across the species, and
efficacious antitumor activity in the multiple preclinical models,
17p was advanced into clinical evaluation.
Select compounds with potent oral activity in the UE model
were then evaluated in an HT1080 fibrosarcoma xenograft tumor
growth model for their antitumor activity. Consistent with their
potent activity in the UE model and good oral exposure profiles,
compounds 17l, 17p, and 22a showed significant tumor growth
inhibition (around 70%) when dosed orally at 10 mg/kg daily
whereas 38 was less efficacious even at a higher dose (30 mg/
kg). Compound 17p was further investigated in a number of
other preclinical tumor growth models and significant efficacy
was also observed. For example, it displayed 87% inhibition of
tumor growth in DLD-1 colon carcinoma and 76% inhibition
in MX-1 breast carcinoma, respectively, at a daily oral dose of
12.5 mg/kg.25
The significant antitumor efficacy of 17p might be ascribed
to its potent multitargeted kinase inhibition. As Table 6 shows,
it potently inhibited all the members of the VEGFR and PDGFR
families, but was generally highly selective over other structur-
ally nonrelated tyrosine kinases, such as SRC and IGFR, as
well as a number of selected serine/threonine kinases (e.g., AKT,
CDC2). As a potent FLT3 inhibitor, 17p may be promising in
treating AMLs with overactivated FLT3. This was demonstrated
by its impressive efficacy in a mouse flank xenograft model of
MV-4-11 leukemia cells, in which FLT3 is constitutively
activated by an ITD mutation. Rapid tumor regression was
achieved when 17p was given twice a day orally at a daily dose
g1.5 mg/kg.25
Conclusion
Potent multitargeted RTK inhibitors with manageable adverse
effects hold great promise as anticancer agents. Through a
structure-based approach, we identified 3-aminoindazole as a
novel kinase inhibitor template, which mimicked the adenine
of ATP interacting with the kinase hinge region through a pair
of hydrogen-bonding interactions. Incorporation of an N,N′-
diaryl urea moiety at the 4-position of the indazole ring afforded
a series of compounds that potently inhibited VEGFR and
PDGFR kinases. A KDR homology model suggested that these
compounds bind to the ATP-binding site of an inactive KDR
conformation, with the urea portion interacting with the distal
hydrophobic pocket. By optimizing the substituents at both the
urea terminal phenyl ring and the 7-position of the 3-aminoin-
dazole, a series of compounds with potent enzymatic and cellular
activity were obtained. A number of these compounds possessed
potent oral activity in the mouse UE model. In particular,
compound 17p was extremely potent, with an ED50 value of
0.5 mg/kg. Further evaluation of 17p showed that it displayed
good PK profiles in different species and significantly inhibited
tumor growth in a number of preclinical animal models. Based
on its overall in vitro and in vivo profile, 17p was chosen for
clinical evaluation.
Experimental Section
Chemistry. 1H NMR spectra were recorded on a 300 MHz
spectrometer (GE QE300) if not otherwise indicated, and chemical
shifts are reported in parts per million (ppm, δ) relative to
tetramethylsilane as an internal standard. Mass spectra were
obtained on a Finnigan MAT SSQ700 instrument. Elemental
analysis (C, H, N) was performed by Robertson Microlit Labora-
tories, Inc., Madison, NJ, and the results indicated by elemental
symbols are within (0.4% of theoretical values. Silica gel 60 (E.
Merck, 230-400 mesh) was used for flash column chromatography.
Preparative HPLC was performed on a Waters Symmetry C8
The efficacy exhibited by 17p in tumor growth inhibition
warranted its further investigation. Although this compound is
physically characterized by high lipophilicity (logD ) 4.2 at
pH 7.4) and low aqueous solubility (27 ng/mL at pH 7 at room
temperature), it displayed favorable PK profiles across the
species. Table 7 shows the PK data obtained from the evaluation
of 17p in Sprague-Dawley rat, Beagle dog, and cynomolgus
monkey. In these species, 17p exhibited moderate volumes of
distribution (1.0-2.2 L/kg) and low plasma clearance (3.3-

1592 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Dai et al.
Figure 6. In vitro metabolic pathways of 17p.
column (25 mm × 100 mm, 7 µm particle size) using a gradient of
10-100% acetonitrile in water containing 0.1% trifluoroacetic
acid (TFA) over 8 min (10 min run time) at a flow rate of 40 mL/
min.
cooled to room temperature, the mixture was partitioned between
ethyl acetate and water. The aqueous layer was extracted twice with
ethyl acetate, and the combined organic layers were washed with
brine, dried (MgSO4), filtered, and concentrated. The crude product
was purified by flash column chromatography on silica gel with
4-Iodo-1H-indazol-3-amine (8). A mixture of 2-fluoro-6-
iodobenzonitrile (2.0 g, 8.1 mmol) and hydrazine monohydrate (4
mL) in n-butanol (40 mL) was stirred at 105-110 °C for 5 h and
then cooled to room temperature, poured into water, and extracted
twice with ethyl acetate. The combined extracts were washed with
water and brine, dried (MgSO4), filtered, and concentrated to
provide the title compound (1.88 g, 90%). 1H NMR (DMSO-d6) δ
5.04 (s, 2H), 6.93 (dd, J ) 8.14, 7.12 Hz, 1H), 7.29 (d, J ) 8.48
Hz, 1H), 7.34 (d, J ) 7.12 Hz, 1H), 11.78 (s, 1H); MS (ESI) m/z
260 (M + H)+.
1
ethyl acetate to give the title compound (60 mg, 59%). H NMR
(DMSO-d6) δ 4.32 (s, 2H), 6.81 (dd, J ) 5.09, 2.71 Hz, 1H), 7.24-
7.33 (m, 2H), 7.47 (d, J ) 8.48 Hz, 2H), 7.51-7.64 (m, 3 H), 7.93
(d, J ) 8.82 Hz, 2H), 7.96-8.01 (m, 2H), 10.39 (s, 1H), 11.72 (s,
1H); MS (ESI) m/z 329 (M + H)+. Anal. (C20H16N4O‚0.3H2O) C,
H, N.
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]benzenesulfonamide
(14). Compound 14 was prepared from 8 using the same procedure
as for 13. 1H NMR (DMSO-d6) δ 4.19 (s, 2H), 6.71 (dd, J ) 4.58,
3.22 Hz, 1H), 7.16-7.26 (m, 4H), 7.29-7.36 (m, 2H), 7.52-7.67
(m, 3H), 7.79 (d, J ) 6.78 Hz, 2H), 10.45 (s, 1H), 11.72 (s, 1H);
MS (ESI) m/z 365 (M + H)+.
4-(4-Aminophenyl)-1H-indazol-3-ylamine (9). A mixture of 8
(467 mg, 1.81 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)aniline (10; 435 mg, 1.99 mmol), Na2CO3 (526 mg, 4.96
mmol), and Pd(PPh3)4 (104 mg, 0.09 mmol) in toluene (20 mL),
ethanol (20 mL), and water (10 mL) was heated at 90 °C for 16 h
under a nitrogen atmosphere. After being cooled to room temper-
ature, the mixture was partitioned between ethyl acetate and water.
The aqueous layer was extracted with ethyl acetate twice, and the
combined organic layers were washed with brine, dried (MgSO4),
filtered, and concentrated. The crude product was purified by flash
column chromatography on silica gel with 3-8% methanol in
General Procedure for the Synthesis of Urea Boronates 16a-
s. A solution of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
aniline (10; 500 mg, 2.28 mmol) in methylene chloride (10 mL)
was treated with the corresponding isocyanate (2.28 mmol) and
stirred at room temperature overnight. The formed solid material
was collected by filtration to provide the desired urea boronates.
In the cases in which only little or no precipitate was formed after
the reaction, the reaction mixture was concentrated and the product
was precipitated out by adding hexanes to the concentrated solution.
N-Phenyl-N′-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
1
dichloromethane to give the title compound (243 mg, 60%). H
NMR (DMSO-d6) δ 4.34 (s, 2H), 5.25 (s, 2H), 6.62-6.74 (m, 3H),
7.06-7.29 (m, 4H), 11.59 (s, 1H); MS (ESI) m/z 225 (M + H)+.
Anal. (C13H12N4‚0.3H2O‚0.1EtOAc) C, H, N.
1
phenyl]urea (16a). H NMR (500 MHz, DMSO-d6) δ 1.28 (s,
12H), 6.98 (t, J ) 7.32 Hz, 1H), 7.26-7.31 (m, 2H), 7.46 (t, J )
8.24 Hz, 4H), 7.59 (d, J ) 8.24 Hz, 2H), 8.70 (s, 1H), 8.81 (s,
1H); MS (ESI) m/z 339 (M + H)+.
N-[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)phenyl]-
benzamide (11). To a solution of 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)aniline (10; 500 mg, 2.28 mmol) in methylene
chloride (10 mL) cooled with an ice-water bath was added
triethylamine (477 µL, 3.42 mmol) and benzoyl chloride (248 µL,
2.28 mmol). The mixture was stirred overnight, washed with water,
dried (MgSO4), filtered, and concentrated to give the title compound
Spectral data of urea boronates 16b-s are given in the Supporting
Information.
General Procedures for the Synthesis of 3-Aminoindazole
Ureas 17a-s. Method A: A mixture of 8 (60 mg, 0.24 mmol),
urea boronate (0.29 mmol), and Na2CO3 (64 mg, 0.60 mmol) under
a nitrogen atmosphere was treated with DME (9 mL), water (3
mL), and Pd(dppf)Cl2‚CH2Cl2 (10 mg, 0.012 mmol). The mixture
was purged with bubbling nitrogen for 2 min, and then stirred at
85 °C for 16 h, cooled to room temperature, and partitioned between
ethyl acetate and water. The aqueous layer was extracted with ethyl
acetate twice, and the combined organic layers were washed with
brine, dried (MgSO4), filtered, and concentrated. The crude product
was purified either by flash column chromatography on silica gel
eluting first with ethyl acetate and then 4-8% methanol in
dichloromethane or by preparative HPLC.
1
as a white solid (700 mg, 95%). H NMR (DMSO-d6) δ 1.30 (s,
12H), 7.50-7.62 (m, 3H), 7.66 (d, J ) 8.48 Hz, 2H), 7.82 (d, J )
8.48 Hz, 2H), 7.93-7.98 (m, 2H), 10.34 (s, 1H); MS (ESI) m/z
324 (M + H)+.
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]benzamide (13). A mix-
ture of 8 (100 mg, 0.31 mmol), 11 (120 mg, 0.37 mmol), Na2CO3
(82 mg, 0.77 mmol), and Pd(dppf)Cl2‚CH2Cl2 (13 mg, 0.015 mmol)
in 1,2-dimethoxyethane (DME; 3 mL) and water (1 mL) was purged
with bubbling nitrogen for 2 min and then heated at 150 °C for 20
min in a Smith Synthesizer microwave oven (300 W). After being

NoVel Tyrosine Kinase ActiVity Inhibitor ABT-869
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1593
Method B: A mixture of 8 (60 mg, 0.24 mmol), urea boronate
(0.29 mmol), Na2CO3 (64 mg, 0.60 mmol), and Pd(dppf)Cl2‚CH2-
Cl2 (10 mg, 0.012 mmol) in DME (3 mL) and water (1 mL) in a
microwave vial was degassed by bubbling through nitrogen for 1-2
min. The reaction vial was then capped and heated at 140-160 °C
under stirring for 15-20 min in a Smith Synthesizer microwave
oven (300 W). The reaction mixture was worked up and purified
as in Method A.
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]-N′-phenylurea (17a).
1H NMR (DMSO-d6) δ 4.33 (s, 2H), 6.78 (dd, J ) 5.42, 2.37 Hz,
1H), 6.98 (t, J ) 7.29 Hz, 1H), 7.28 (m, 4H), 7.39 (d, J ) 8.81
Hz, 2H), 7.48 (d, J ) 7.46 Hz, 2H), 7.59 (d, J ) 8.82 Hz, 2H),
8.72 (s, 1H), 8.81 (s, 1H), 11.70 (s, 1H); MS (ESI) m/z 344 (M +
H)+. Anal. (C20H17N5O‚0.5EtOAc) C, H, N.
1H), 8.75 (s, 1H), 11.69 (s, 1H); MS (ESI) m/z 358 (M + H)+.
Anal. (C21H19N5O‚0.4H2O) C, H, N.
N-(4-(3-Amino-1H-indazol-4-yl)phenyl)-N′-(3-ethylphenyl)-
1
urea (17k). H NMR (DMSO-d6) δ 1.19 (t, J ) 7.63 Hz, 3H),
2.58 (q, J ) 7.68 Hz, 2H), 6.80-6.89 (m, 2H), 7.19 (t, J ) 7.80
Hz, 1H), 7.25-7.45 (m, 6H), 7.60 (d, J ) 8.81 Hz, 2H), 8.70 (s,
1H), 8.85 (s, 1H); MS (ESI) m/z 372 (M + H)+. Anal.
(C22H21N5O‚0.8CF3CO2H‚0.2H2O) C, H, N.
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]-N′-(3-chlorophenyl)-
1
urea (17l). H NMR (DMSO-d6) δ 6.89 (dd, J ) 6.44, 1.70 Hz,
1H), 7.03 (m, 1H), 7.28-7.38 (m, 4H), 7.42 (d, J ) 8.81 Hz, 2H),
7.61 (d, J ) 8.82 Hz, 2H), 7.74 (m, 1H), 9.00 (s, 1H), 9.03 (s,
1H); MS (ESI) m/z 378 (M + H)+. Anal. (C20H16ClN5O‚1.0CF3-
CO2H‚0.5H2O) C, H, N.
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]-N′-(3-bromophenyl)-
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]-N′-(3-methylphenyl)-
1
1
urea (17m). H NMR (DMSO-d6) δ 6.87 (dd, J ) 6.10, 1.70 Hz,
urea (17b). H NMR (DMSO-d6) δ 2.29 (s, 3H), 4.33 (s, 2H),
1H), 7.13-7.18 (m, 1H), 7.25 (t, J ) 7.97 Hz, 1H), 7.31-7.26
(m, 3H), 7.42 (d, J ) 8.82 Hz, 2H), 7.61 (d, J ) 8.48 Hz, 2H),
7.88 (t, J ) 1.86 Hz, 1H), 8.99 (s, 1H); MS (ESI) m/z 420, 422 (M
- H)-. Anal. (C20H16BrN5O‚0.1C6H14‚0.1CH2Cl2) C, H, N.
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]-N′-[3-(trifluorometh-
yl)phenyl]urea (17n). 1H NMR (DMSO-d6) δ 6.86 (dd, J ) 6.10,
2.03 Hz, 1H), 7.29-7.35 (m, 3H), 7.42 (d, J ) 8.81 Hz, 2H), 7.53
(t, J ) 7.97 Hz, 1H), 7.58-7.65 (m, 3H), 8.05 (s, 1H), 9.01 (s,
1H), 9.16 (s, 1H). MS (ESI) m/z 412 (M + H)+. Anal.
(C21H16F3N5O‚0.7CF3CO2H) C, H, N.
6.76-6.83 (m, 2H), 7.17 (t, J ) 7.80 Hz, 1H), 7.23-7.28 (m, 3H),
7.32 (s, 1H), 7.39 (d, J ) 8.48 Hz, 2H), 7.59 (d, J ) 8.48 Hz, 2H),
8.64 (s, 1H), 8.79 (s, 1H), 11.70 (s, 1H); MS (ESI) m/z 358 (M +
H)+. Anal. (C21H19N5O‚0.8H2O) C, H, N.
N-[4-(3-Amino-1H-indazol-4-yl)-phenyl]-N′-thiophen-3-yl-
1
urea (17c). H NMR (DMSO-d6) δ 4.33 (s, 2H), 6.78 (dd, J )
5.59, 2.20 Hz, 1H), 7.07 (dd, J ) 5.09, 1.36 Hz, 1H), 7.24-7.28
(m, 2H), 7.29-7.33 (m, 1H), 7.39 (d, J ) 8.48 Hz, 2H), 7.45 (dd,
J ) 5.09, 3.39 Hz, 1H), 7.59 (d, J ) 8.48 Hz, 2H), 8.78 (s, 1H),
8.99 (s, 1H), 11.70 (s, 1H); MS (ESI) m/z 350 (M + H)+. Anal.
(C18H15N5OS‚0.1C6H14•0.3H2O) C, H, N.
N-(4-(3-Amino-1H-indazol-4-yl)phenyl)-N′-(3-hydroxypheny-
1
l)urea (17o). H NMR (DMSO-d6) δ 5.33 (s, 2H), 6.38 (dd, J )
N-[4-(3-Amino-1H-indazol-4-yl)-phenyl]-N′-cyclopentylurea
(17d). 1H NMR (DMSO-d6) δ 1.31-1.45 (m, 2 H), 1.47-1.71 (m,
4H), 1.77-1.93 (m, 2H), 3.89-4.00 (m, 1H), 4.31 (s, 2H), 6.21
(d, J ) 7.12 Hz, 1H), 6.75 (dd, J ) 5.59, 2.20 Hz, 1H), 7.22-7.27
(m, 2H), 7.32 (d, J ) 8.48 Hz, 2H), 7.50 (d, J ) 8.48 Hz, 2H),
8.39 (s, 1H), 11.68 (s, 1H); MS (ESI) m/z 336 (M + H)+.
N-[4-(3-Amino-1H-indazol-4-yl)-phenyl]-N′-cyclohexylurea
(17e). 1H NMR (DMSO-d6) δ 1.09-1.41 (m, 5H), 1.48-1.60 (m,
1H), 1.60-1.73 (m, 2H), 1.77-1.89 (m, 2H), 3.42-3.56 (m, 1H),
4.30 (s, 2H), 6.12 (d, J ) 7.80 Hz, 1H), 6.75 (dd, J ) 5.59, 2.20
Hz, 1H), 7.19-7.26 (m, 2H), 7.32 (d, J ) 8.82 Hz, 2H), 7.50 (d,
J ) 8.82 Hz, 2H), 8.43 (s, 1H), 11.67 (s, 1H); MS (ESI) m/z 350
(M + H)+. Anal. (C20H23N5O‚0.2H2O‚0.3EtOAc) C, H, N.
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]-N′-(2-fluorophenyl)-
7.63, 1.86 Hz, 1H), 6.76-6.92 (m, 2H), 6.99-7.14 (m, 2 H), 7.26-
7.45 (m, 4 H), 7.59 (d, J ) 8.81 Hz, 2H), 8.65 (s, 1H), 8.80 (s,
1H), 9.31 (s, 1H), 12.11 (s, 1H); MS (ESI) m/z 360 (M + H). Anal.
(C20H17N5O2‚0.85CF3CO3H) C, H, N.
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]-N′-(2-fluoro-5-meth-
ylphenyl)urea (17p). 1H NMR (DMSO-d6) δ 2.28 (s, 3H), 6.78-
6.85 (m, 1H), 6.87 (dd, J ) 6.10, 1.70 Hz, 1H), 7.12 (dd, J )
11.53, 8.48 Hz, 1H), 7.30-7.39 (m, 2H), 7.42 (d, J ) 8.81 Hz,
2H), 7.60 (d, J ) 8.48 Hz, 2H), 8.01 (dd, J ) 7.80, 2.37 Hz, 1H),
8.54 (d, J ) 2.71 Hz, 1H), 9.23 (s, 1H); MS (ESI) m/z 376 (M +
H)+. Anal. (C21H18FN5O‚0.8CF3CO2H) C, H, N.
N-(4-(3-Amino-1H-indazol-4-yl)phenyl)-N′-(4-fluoro-3-meth-
1
ylphenyl)urea (17q). H NMR (DMSO-d6) δ 2.22 (d, J ) 1.70
Hz, 3H), 6.84 (dd, J ) 6.10, 1.70 Hz, 1H), 7.06 (t, J ) 9.16 Hz,
1H), 7.24-7.45 (m, 6 H), 7.59 (d, J ) 8.48 Hz, 2H), 8.69 (s, 1H),
8.84 (s, 1H); MS (ESI) m/z 376 (M + H)+. Anal. (C21H18-
FN5O‚0.8CF3CO3H) C, H, N.
1
urea (17f). H NMR (DMSO-d6) δ 4.33 (s, 2H), 6.79 (dd, J )
5.42, 2.71 Hz, 1H), 6.97-7.06 (m, 1H), 7.16 (t, J ) 7.63 Hz, 1H),
7.22-7.30 (m, 3H), 7.41 (d, J ) 8.48 Hz, 2H), 7.60 (d, J ) 8.48
Hz, 2H), 8.18 (m, 1H), 8.61 (d, J ) 2.37 Hz, 1H), 9.22 (s, 1H),
11.71 (s, 1H); MS (ESI) m/z 362 (M + H)+. Anal. (C20H16-
FN5O‚0.7CH2Cl2‚0.1EtOAc) C, H, N.
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]-N′-(3-fluoro-4-meth-
ylphenyl)urea (17r). 1H NMR (DMSO-d6) δ 2.17 (s, 3H), 4.33 (s,
2H), 6.78 (dd, J ) 5.42, 2.71 Hz, 1H), 7.05 (dd, J ) 8.14, 2.03
Hz, 1H), 7.17 (t, J ) 8.65 Hz, 1H), 7.23-7.30 (m, 2H), 7.39 (d, J
) 8.48 Hz, 2H), 7.45 (dd, J ) 12.54, 2.03 Hz, 1H), 7.58 (d, J )
8.81 Hz, 2H), 8.84 (s, 2H), 11.70 (s, 1H); MS (ESI) m/z 376 (M +
H)+. Anal. (C21H18FN5O‚0.6CF3CO3H) C, H, N.
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]-N′-(3-fluorophenyl)-
1
urea (17g). H NMR (DMSO-d6) δ 4.33 (s, 2H), 6.75-6.83 (m,
2H), 7.15 (m, 1H), 7.23-7.36 (m, 3H), 7.40 (d, J ) 8.48 Hz, 2H),
7.52 (m, 1H), 7.59 (d, J ) 8.48 Hz, 2H), 8.89 (s, 1H), 8.97 (s,
1H), 11.71 (s, 1H); MS (ESI) m/z 362 (M + H)+. Anal. (C20H16-
FN5O‚0.2C6H14‚0.2EtOAc) C, H, N.
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]-N′-[2-fluoro-5-(trif-
1
luoromethyl)phenyl]urea (17s). H NMR (DMSO-d6) δ 4.33 (s,
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]-N′-(4-fluorophenyl)-
2H), 6.79 (dd, J ) 5.26, 2.54 Hz, 1H), 7.24-7.31 (m, 2H), 7.42
(m, 3H), 7.52 (m, 1H), 7.61 (d, J ) 8.48 Hz, 2H), 8.65 (dd, J )
7.29, 2.20 Hz, 1H), 8.96 (d, J ) 3.05 Hz, 1H), 9.32 (s, 1H), 11.72
(s, 1H); MS (ESI) m/z 430 (M + H)+.
1
urea (17h). H NMR (DMSO-d6) δ 4.33 (s, 2H), 6.78 (dd, J )
5.43, 2.37 Hz, 1H), 7.13 (t, J ) 8.99 Hz, 2H), 7.22-7.29 (m, 2H),
7.39 (d, J ) 8.48 Hz, 2H), 7.49 (m, 2H), 7.59 (d, J ) 8.82 Hz,
2H), 8.80 (d, J ) 15.60 Hz, 2H), 11.70 (s, 1H); MS (ESI) m/z 362
(M + H)+. Anal. (C20H16FN5O‚0.7H2O) C, H, N.
4-Iodo-1-methyl-1H-indazol-3-amine (20a). A mixture of
2-fluoro-6-iodobenzonitrile (7; 2.6 g, 10.5 mmol) and methyl
hydrazine (5.7 mL, 105 mmol) in n-butanol (60 mL) was stirred at
100-110 °C for 3 h and then cooled to room temperature and
concentrated. The residue was partitioned between water and ethyl
acetate. The aqueous layer was extracted twice with ethyl acetate.
The combined extracts were washed with water and brine, dried
(MgSO4), filtered, and concentrated to provide the title compound
N-[4-(3-Amino-1H-indazol-4-yl)phenyl]-N′-(2-methylphenyl)-
1
urea (17i). H NMR (DMSO-d6) δ 2.21-2.33 (m, 3H), 4.33 (s,
2H), 6.78 (dd, J ) 5.76, 2.37 Hz, 1H), 6.96 (t, J ) 7.46 Hz, 1H),
7.12-7.21 (m, 2H), 7.22-7.32 (m, 2H), 7.39 (d, J ) 8.82 Hz,
2H), 7.60 (d, J ) 8.82 Hz, 2H), 7.84 (d, J ) 7.12 Hz, 1H), 7.97 (s,
1H), 9.15 (s, 1H), 11.69 (s, 1H); MS (ESI) m/z 358 (M + H)+.
Anal. (C21H19N5O‚0.6H2O) C, H, N.
1
(2.5 g, 87%). H NMR (DMSO-d6) δ 3.74 (s, 3H), 5.11 (s, 2H),
6.97 (dd, J ) 8.48, 7.46 Hz, 1H), 7.35 (d, J ) 7.46 Hz, 1H), 7.40
N-[4-(3-Amino-1H-indazol-4-yl)-phenyl]-N′-(4-methylphenyl)-
urea (17j). 1H NMR (DMSO-d6) δ 2.25 (s, 3H), 4.32 (s, 2H), 6.78
(dd, J ) 5.43, 2.37 Hz, 1H), 7.10 (d, J ) 8.14 Hz, 2H), 7.21-7.31
(m, 2H), 7.33-7.43 (m, 4H), 7.58 (d, J ) 8.82 Hz, 2H), 8.60 (s,
(d, J ) 8.48 Hz, 1H); MS (ESI) m/z 274 (M + H)+.
2-(4-Iodo-1H-indazol-3-yl)-1H-isoindole-1,3(2H)-dione (18). A
mixture of 8 (1.09 g, 4.21 mmol) and phthalic anhydride (0.75 g,

1594 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Dai et al.
5.26 mmol) in dioxane (15 mL) was stirred at 110 °C overnight
and then concentrated. The residue was triturated from diethyl ether
(15 mL) to provide the title compound (0.51 g, 31%). MS (ESI)
m/z 388 (M + H)+.
2-{4-Iodo-1-[2-(4-morpholinyl)ethyl]-1H-indazol-3-yl}-1H-
isoindole-1,3(2H)-dione (19c). A mixture of 18 (100 mg, 0.26
mmol), 4-(2-chloroethyl)morpholine (48 mg, 0.32 mmol), and Na2-
CO3 (82 mg, 0.77 mmol) in DMF (5 mL) was heated overnight at
80 °C, cooled to room temperature, and partitioned between 1 N
HCl (aq) and ethyl acetate. The aqueous layer was separated,
basified with 1 N KOH (aq), and extracted twice with ethyl acetate.
The combined extracts were washed with water and brine, dried
(MgSO4), filtered, and concentrated to provide the title compound
(45 mg, 34%). MS (ESI) m/z 503 (M + H)+.
Hz, 1H), 6.87 (d, J ) 6.86 Hz, 1H), 7.17 (t, J ) 7.80 Hz, 1H),
7.26 (d, J ) 8.42 Hz, 1H), 7.32 (s, 1H), 7.37-7.41 (m, 3H), 7.48
(d, J ) 8.11 Hz, 1H), 7.61 (d, J ) 8.42 Hz, 2H), 8.75 (s, 1H), 8.93
(s, 1H); MS (ESI) m/z 471 (M + H)+. Anal. (C27H30N6O2‚2.0CF3-
CO2H) C, H, N.
N-{4-[3-Amino-1-(2-methoxyethyl)-1H-indazol-4-yl]phenyl}-
N′-(2-fluoro-5-methylphenyl)urea (22d). Compound 22d was
1
prepared from 18 using the same procedures as for 22c. H NMR
(DMSO-d6) δ 2.28 (s, 3H), 3.21 (s, 3H), 3.69 (t, J ) 5.43 Hz, 2H),
4.32 (t, J ) 5.43 Hz, 2H), 6.77-6.86 (m, 2H), 7.12 (dd, J ) 11.36,
8.31 Hz, 1H), 7.23-7.35 (m, 1H), 7.40 (d, J ) 8.48 Hz, 2H), 7.59
(d, J ) 8.48 Hz, 2H), 8.01 (dd, J ) 7.80, 2.03 Hz, 1H), 8.54 (d, J
) 2.37 Hz, 1H), 9.21 (s, 1H); MS (ESI) m/z 434 (M + H)+. Anal.
(C24H24N5O2‚0.5CF3CO2H‚0.5H2O) C, H, N.
4-Iodo-1-[2-(4-morpholinyl)ethyl]-1H-indazol-3-amine (20c).
A mixture of hydrazine monohydrate (58 µL, 1.2 mmol) and 19c
(120 mg, 0.24 mmol) in ethanol (5 mL) was stirred at 0 °C for 1
h and then at room temperature for 3 h. The mixture was
concentrated. The residue was purified by flash column chroma-
tography on silica gel with 5-8% methanol in dichloromethane to
provide the title compound (80 mg, 90%). MS (ESI) 373 (M +
H)+.
2-Fluoro-6-iodo-3-methylbenzoic acid (24a). A solution of
2-fluoro-4-iodo-1-methylbenzene (23a; 25 g, 105.9 mmol) in THF
(200 mL) was treated dropwise with LDA (2 M solution in THF,
58.5 mL, 116 mmol) at -78 °C. After being stirred at -78 °C for
1 h, the reaction mixture was treated with excess solid carbon
monoxide, and stirred overnight while the reaction temperature
slowly rose to room temperature. The mixture was concentrated
and then partitioned between 4 N NaOH (aq) and diethyl ether.
The aqueous phase was adjusted to pH 2 with 2 N HCl (aq) and
then extracted three times with ethyl acetate. The combined extracts
were washed with water and brine, dried (MgSO4), filtered, and
concentrated to provide the title compound (19.4 g, 66%). 1H NMR
(DMSO-d6) δ 2.21 (d, J ) 2.03 Hz, 3H), 7.14 (t, J ) 7.97 Hz,
1H), 7.59 (d, J ) 8.14 Hz, 1H), 13.84 (s, 1H); MS (ESI) m/z 279
(M + H)+.
4-(4-Aminophenyl)-1-methyl-1H-indazol-3-amine (21a). A
mixture of 20a (2.50 g, 9.16 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)aniline (10; 3.0 g, 13.7 mmol), Pd(PPh3)4 (528
mg, 0.46 mmol), and Na2CO3 (2.9 g, 27.48 mmol) in 1,2-
dimethoxyethane (40 mL) and water (10 mL) was heated at 90 °C
under a nitrogen atmosphere for 16 h. The mixture was concentrated
and partitioned between ethyl acetate and water. The aqueous phase
was extracted with ethyl acetate twice. The combined organic layers
were washed with brine, dried (MgSO4), filtered, and concentrated.
The crude product was purified by flash column chromatography
on silica gel with 0-5% methanol in dichloromethane to provide
2-Fluoro-6-iodo-3-methylbenzamide (25a). A mixture of 24a
(19.3 g, 69.1 mmol) in thionyl chloride (60 mL) was heated at 80
°C for 3 h, cooled to room temperature, and concentrated. The
residue was dissolved in THF (100 mL), treated at 0 °C with
concentrated NH4OH (aq; 80 mL), stirred at room temperature
overnight, and concentrated. The residue was suspended in water
and filtered. The filter cake was washed with water and dried to
1
the title compound (1.4 g, 65%). H NMR (DMSO-d6) δ 3.76 (s,
1H), 4.40 (s, 2H), 5.27 (s, 2H), 6.64-6.73 (m, 3H), 7.12 (d, J )
8.14 Hz, 2H), 7.24-7.27 (m, 2H); MS (ESI) m/z 239 (M + H)+.
N-[4-(3-Amino-1-methyl-1H-indazol-4-yl)-phenyl]-N′-(3-me-
thylphenyl)urea (22a). To a solution of 21a (80 mg, 0.34 mmol)
in DMF (3 mL) at 0 °C was added m-tolyl isocyanate (43 µL, 0.34
mmol). The mixture was stirred overnight and then partitioned
between ethyl acetate. The aqueous phase was extracted with ethyl
acetate twice. The combined organic phases were washed water
and brine, dried (MgSO4), filtered, and concentrated. The crude
product was purified by flash column chromatography on silica
gel with 0-3% methanol in dichloromethane to give the title
1
provide the title compound (18.67 g, 97%). H NMR (DMSO-d6)
δ 2.20 (d, J ) 2.03 Hz, 3H), 7.07 (t, J ) 8.14 Hz, 1H), 7.55 (d, J
) 8.14 Hz, 1H), 7.70 (s, 1H), 7.97 (s, 1H); MS (CI) m/z 280 (M
+ H)+.
2-Fluoro-6-iodo-3-methylbenzonitrile (26a). A solution of 25a
(18.6 g, 66.7 mmol) in DMF (190 mL) was treated dropwise with
thionyl chloride (24 mL, 333 mmol), heated at 115 °C for 16 h,
cooled to room temperature, poured into ice, and extracted three
times with ethyl acetate. The combined extracts were washed with
water and brine, dried (MgSO4), filtered, and concentrated. The
residue was purified by flash column chromatography on silica gel
with 25% ethyl acetate in hexanes to provide the title compound
(12.35 g, 71%). 1H NMR (DMSO-d6) δ 2.24 (d, J ) 2.03 Hz, 3H),
7.44 (t, J ) 7.97 Hz, 1H), 7.78 (d, J ) 8.14 Hz, 1H); MS (CI) m/z
279 (M + NH4)+.
4-Iodo-7-methyl-1H-indazol-3-amine (27a). Compound 27a
was prepared from 26a using the same procedure as for 8. 1H NMR
(DMSO-d6) δ 2.33 (s, 3H), 5.02 (s, 2H), 6.74 (d, J ) 7.46 Hz,
1H), 7.24 (d, J ) 7.12 Hz, 1H), 11.83 (s, 1H); MS (ESI) m/z 273.8
(M + H)+.
1
compound (40 mg, 31%). H NMR (DMSO-d6) δ 2.29 (s, 3H),
3.79 (s, 3H), 4.39 (s, 2H), 6.79 (dd, J ) 6.10, 1.70 Hz, 2H), 7.17
(t, J ) 7.80 Hz, 1H), 7.24-7.40 (m, 6H), 7.59 (d, J ) 8.48 Hz,
2H), 8.64 (s, 1H), 8.80 (s, 1H); MS (ESI) m/z 372 (M + H)+. Anal.
(C22H21N5O‚0.1H2O) C, H, N.
N-{4-[3-Amino-1-(2-hydroxyethyl)-1H-indazol-4-yl]phenyl}-
N′-(3-methylphenyl)urea (22b). A mixture of 20b (50 mg, 0.166
mmol), 16b (75 mg, 0.216 mmol), Pd(PPh3)4 (19 mg, 0.017 mmol),
and Na2CO3 (52 mg, 0.498 mmol) in 1,2-dimethoxyethane (3 mL)
and water (1 mL) was heated at 90 °C under a nitrogen atmosphere
for 16 h. The mixture was concentrated and partitioned between
ethyl acetate and water. The aqueous phase was extracted with ethyl
acetate twice. The combined organic layers were washed with brine,
dried (MgSO4), filtered, and concentrated. The crude product was
purified by preparative HPLC to provide the title compound (26
4-(4-Aminophenyl)-7-methyl-1H-indazol-3-amine (28a). Com-
pound 28a was prepared from 27a and 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)aniline (10) using the same procedure as for 9.
1H NMR (DMSO-d6) δ 2.39 (s, 3H), 4.33 (s, 2H), 5.21 (s, 2H),
6.60 (d, J ) 6.78 Hz, 1H), 6.99 (d, J ) 7.80 Hz, 1H), 7.09 (d, J )
8.48 Hz, 2H), 11.62 (s, 1H); MS (ESI) m/z 239 (M + H)+.
1
mg, 39%). H NMR (DMSO-d6) δ 2.29 (s, 3H), 3.74 (t, J ) 5.76
Hz, 2H), 4.20 (t, J ) 5.59 Hz, 2H), 6.79 (d, J ) 6.78 Hz, 1H),
6.80 (d, J ) 7.12 Hz, 1H), 7.17 (t, J ) 7.63 Hz, 1H), 7.23-7.40
(m, 6H), 7.59 (d, J ) 8.81 Hz, 2H), 8.65 (s, 1H), 8.80 (s, 1H); MS
(ESI) m/z 402 (M + H)+. Anal. (C23H23N5O2‚0.7CF3CO2H) C, H,
N.
N-[4-(3-Amino-7-methyl-1H-indazol-4-yl)phenyl]-N′-(3-meth-
ylphenyl)urea (29a). Compound 29a was prepared from 28a and
1
m-tolyl isocyanate using the same procedure as for 22a. H NMR
N-{4-[3-Amino-1-(2-morpholin-4-yl-ethyl)-1H-indazol-4-yl]-
phenyl}-N′-(3-methylphenyl)urea (22c). Compound 22c was
prepared from 20c using the same procedures as for 22a. 1H NMR
(500 MHz, DMSO-d6) δ 2.29 (s, 3H), 3.58 (t, J ) 5.77 Hz, 2H),
3.39-3.88 (m, 8H), 4.55 (t, J ) 6.39 Hz, 2H), 6.80 (d, J ) 7.49
(DMSO-d6) δ 2.29 (s, 3H), 2.42 (s, 3H), 6.70 (d, J ) 7.1 Hz, 1H),
6.80 (d, J ) 7.5 Hz, 1H), 7.05 (dd, J ) 7.1, 1.0 Hz, 1H), 7.16 (t,
J ) 7.8 Hz, 1H), 7.25 (d, J ) 8.5 Hz, 1H), 7.32 (s, 1H), 7.36 (d,
J ) 8.5 Hz, 2H), 7.57 (d, J ) 8.5 Hz, 2H), 8.63 (s, 1H), 8.77 (s,
1H), 11.75 (s, 1H); MS (ESI) m/z 372 (M + H)+.

NoVel Tyrosine Kinase ActiVity Inhibitor ABT-869
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1595
N-[4-(3-Amino-7-methoxy-1H-indazol-4-yl)phenyl]-N′-(3-me-
thylphenyl)urea (29b). Compound 29b was prepared from 23b
using the same procedures as for 29a. 1H NMR (DMSO-d6) δ 2.29
(s, 3H), 3.92 (s, 3H), 4.30 (s, 2H), 6.69 (d, J ) 7.80 Hz, 1H), 6.78
(d, J ) 7.79 Hz, 1H), 6.80 (d, J ) 7.46 Hz, 1H), 7.16 (t, J ) 7.80
Hz, 1H), 7.25 (m, 1H), 7.31 (s, 1H), 7.34 (d, J ) 8.48 Hz, 2H),
7.55 (d, J ) 8.82 Hz, 2H), 8.62 (s, 1H), 8.75 (s, 1H), 11.86 (s,
1H); MS (ESI) m/z 388 (M + H)+. Anal. (C22H21N5O20‚0.4H2O)
C, H, N.
4-(4-Aminophenyl)-7-(2-dimethylaminoethoxy)-1H-indazol-3-
ylamine (33a). A mixture of 32a (468 mg, 1.35 mmol), 10 (326
mg, 1.68 mmol), Pd(PPh3)4 (50 mg, 0.043 mmol), and Na2CO3 (428
mg, 4.05 mmol) in DME (9 mL) and H2O (3 mL) was heated at 85
°C under a nitrogen atmosphere for 16 h and then partitioned
between ethyl acetate and water. The aqueous phase was extracted
twice with ethyl acetate. The combined organic phases were washed
with water and brine, dried (MgSO4), filtered, and concentrated.
The residue was purified by flash column chromatography on silica
gel eluting with 12% methanol in dichloromethane to provide the
N-[4-(3-Amino-7-fluoro-1H-indazol-4-yl)phenyl]-N′-(3-meth-
ylphenyl)urea (29c). Compound 29c was prepared from 23c using
1
title compound (234 mg, 56%). H NMR (DMSO-d6) δ 2.25 (s,
1
6H), 2.71 (t, J ) 5.76 Hz, 2H), 4.20 (t, J ) 5.93 Hz, 2H), 4.30 (s,
2H), 5.17 (s, 2H), 6.58 (d, J ) 7.80 Hz, 1H), 6.65 (d, J ) 8.48 Hz,
2H), 6.74 (d, J ) 7.80 Hz, 1H), 7.07 (d, J ) 8.14 Hz, 2H), 11.68
(s, 1H); MS (ESI) m/z 312 (M + H)+.
the same procedures as for 29a. H NMR (DMSO-d6) δ 2.29 (s,
3H), 6.75 (dd, J ) 7.80, 4.41 Hz, 1H), 6.80 (d, J ) 7.46 Hz, 1H),
7.15 (m, 2H), 7.25 (m, 1H), 7.32 (s, 1H), 7.37 (d, J ) 8.48 Hz,
2H), 7.59 (d, J ) 8.48 Hz, 2H), 8.66 (s, 1H), 8.82 (s, 1H); MS
(ESI) m/z 376 (M + H)+. Anal. (C21H18FN5O‚0.4H2O) C, H, N.
N-[4-(3-Amino-7-bromo-1H-indazol-4-yl)phenyl]-N′-(3-meth-
ylphenyl)urea (29d). Compound 29d was prepared from 23d using
N-{4-[3-Amino-7-(2-dimethylamino-ethoxy)-1H-indazol-4-yl]-
phenyl}-N′-(3-methylphenyl)urea (34a). Compound 34a was
prepared from 33a and m-tolyl isocyanate using the same procedure
as for 22a. 1H NMR (500 MHz, DMSO-d6) δ 2.29 (s, 3H), 2.95 (s,
6H), 3.61 (br s, 2H), 4.50 (m, 2H), 6.77 (d, J ) 7.63 Hz, 1H), 6.80
(d, J ) 7.32 Hz, 1H), 6.90 (d, J ) 7.63 Hz, 1H), 7.16 (t, J ) 7.78
Hz, 1H), 7.26 (d, J ) 8.54 Hz, 1H), 7.33 (s, 1H), 7.36 (d, J ) 8.54
Hz, 2H), 7.59 (d, J ) 8.54 Hz, 2H), 8.81 (s, 1 H), 8.97 (s, 1H);
MS (ESI) MS m/z 445 (M + H)+. Anal. (C25H28N6O2‚1.8CF3CO2H)
C, H, N.
1
the same procedures as for 29a. H NMR (DMSO-d6) δ 2.29 (s,
3H), 4.46 (s, 2H), 6.73 (d, J ) 7.46 Hz, 1H), 6.80 (d, J ) 6.78 Hz,
1H), 7.17 (t, J ) 7.63 Hz, 1H), 7.24-7.32 (m, 2H), 7.39 (d, J )
8.48 Hz, 2H), 7.50 (d, J ) 7.80 Hz, 1H), 7.60 (d, J ) 8.14 Hz,
2H), 8.64 (s, 1H), 8.82 (s, 1H), 12.08 (s, 1H); MS (ESI) m/z 434,
436 (M - H)-. Anal. (C21H18BrN5O) C, H, N.
2-Fluoro-6-iodo-3-methoxybenzonitrile (26b). Compound 26b
1
N-{4-[3-Amino-7-(2-diethylaminoethoxy)-1H-indazol-4-yl]-
phenyl}-N′-(3-methylphenyl)urea (34b). Compound 34b was
was prepared from 23b using the same procedures as for 26a. H
NMR (DMSO-d6) δ 3.89 (s, 3H), 7.36 (t, J ) 8.82 Hz, 1H), 7.80
(dd, J ) 8.82, 1.36 Hz, 1H); MS (ESI) m/z 278 (M + H)+.
2-Fluoro-3-hydroxy-6-iodobenzonitrile (30). A solution of 26b
(148 mg, 0.54 mmol) in dichloromethane (5 mL) was treated at
-78 °C dropwise with BBr3 (2.5 mL, 1 M in dichloromethane, 2.5
mmol), warmed to room temperature, stirred for 18 h, poured into
water, and extracted with diethyl ether. The extract was dried
(MgSO4), filtered, and concentrated. The residue was purified by
flash column chromatography on silica gel with 20% ethyl acetate
in hexanes to provide the title compound (110 mg, 77%). 1H NMR
(DMSO-d6) 7.09 (t, J ) 8.82 Hz, 1H), 7.62 (dd, J ) 8.65, 1.53
Hz, 1H), 10.96 (s, 1H); MS (ESI) m/z 262 (M - H)-.
1
prepared from 30 using the same procedures as for 34a. H NMR
(500 MHz, DMSO-d6) δ 1.27 (t, J ) 7.33 Hz, 6H), 2.29 (s, 3H),
3.34 (br s, 4H), 3.61 (br s, 2H), 4.49 (t, J ) 4.80 Hz, 2H), 6.74 (d,
J ) 7.49 Hz, 1H), 6.80 (d, J ) 7.17 Hz, 1H), 6.88 (d, J ) 7.80
Hz, 1H), 7.16 (t, J ) 7.80 Hz, 1H), 7.26 (d, J ) 8.11 Hz, 1H),
7.32 (s, 1H), 7.35 (d, J ) 8.42 Hz, 2H), 7.58 (d, J ) 8.42 Hz, 2H),
8.72 (s, 1H), 8.87 (s, 1H); MS (ESI) m/z 473 (M + H)+. Anal.
(C27H32N6O2‚2.0CF3CO2H) C, H, N.
N-{4-[3-Amino-7-(2-pyrrolidin-1-yl-ethoxy)-1H-indazol-4-yl]-
phenyl}-N′-(3-methylphenyl)urea (34c). Compound 34c was
1
prepared from 30 using the same procedures as for 34a. H NMR
(500 MHz, DMSO-d6) δ 1.93 (br s, 2H), 2.07 (br s, 2H), 2.29 (s,
3H), 3.25 (br s, 2H), 3.69 (br s, 4H), 4.48 (t, J ) 4.80 Hz, 2H),
6.77 (d, J ) 7.80 Hz, 1H), 6.80 (d, J ) 7.49 Hz, 1H), 6.90 (d, J )
7.80 Hz, 1H), 7.16 (t, J ) 7.80 Hz, 1H), 7.26 (d, J ) 8.11 Hz,
1H), 7.33 (s, 1H), 7.36 (d, J ) 8.73 Hz, 2H), 7.59 (d, J ) 8.73 Hz,
2H), 8.79 (s, 1H), 8.95 (s, 1H); MS (ESI) m/z 471 (M + H)+. Anal.
(C27H30N6O2‚1.8CF3CO2H) C, H, N.
3-(2-Dimethylaminoethoxy)-2-fluoro-6-iodobenzonitrile (31a).
To a mixture of 30 (830 mg, 3.16 mmol), 2-dimethylaminoethanol
(0.317 mL, 3.79 mmol), and PPh3 on resin (3 mmol/g, 2.11 g, 6.32
mmol) in THF (30 mL) at 0 °C was dropwise added diethyl
azodicarboxylate (DEAD; 600 µL, 3.79 mmol). The mixture was
stirred overnight, while the temperature slowly rose to room
temperature, and then filtered. The residue was purified by flash
column chromatography on silica gel eluting first with 75% ethyl
acetate in hexane and then with 10% methanol in dichloromethane
N-{4-[3-Amino-7-(2-morpholin-4-ylethoxy)-1H-indazol-4-yl]-
phenyl}-N′-(3-methylphenyl)urea (34d). Compound 34d was
1
1
to provide the title compound (834 mg, 79%). H NMR (DMSO-
prepared from 30 using the same procedures as for 34a. H NMR
d6) δ 2.20 (s, 6H), 2.63 (t, J ) 5.59 Hz, 2H), 4.18 (t, J ) 5.76 Hz,
2H), 7.39 (t, J ) 8.82 Hz, 1H), 7.77 (dd, J ) 8.82, 1.70 Hz, 1H);
MS (ESI) m/z 335 (M + H)+.
(DMSO-d6) δ 2.29 (s, 3H), 2.52-2.55 (m, 4H), 2.79 (t, J ) 5.76
Hz, 2H), 3.57-3.60 (m, 4H), 4.26 (t, J ) 5.76 Hz, 2H), 4.30 (s,
2H), 6.68 (d, J ) 7.46 Hz, 1H), 6.80 (d, J ) 7.80 Hz, 2H), 7.16 (t,
J ) 7.80 Hz, 1H), 7.25 (m, 1H), 7.31 (s, 1H), 7.34 (d, J ) 8.48
Hz, 2H), 7.55 (d, J ) 8.82 Hz, 2H), 8.64 (s, 1H), 8.77 (s, 1H),
11.81 (s, 1 H); MS (ESI) MS m/z 487 (M + H)+. Anal.
(C27H30N6O3‚0.3H2O) C, H, N.
2-Fluoro-6-iodo-3-(2-methoxyethoxy)benzonitrile (31f). A mix-
ture of 30 (104 mg, 0.39 mmol), 1-bromo-2-methoxyethane (88
µL, 0.94 mmol), and K2CO3 (163 mg, 1.54 mmol) in acetone (5
mL) was heated at 60 °C for 18 h, cooled to room temperature,
and partitioned between diethyl ether and water. The extract was
dried (MgSO4), filtered, and concentrated to provide the title
N-(4-{3-Amino-7-[2-(2-oxopyrrolidin-1-yl)ethoxy]-1H-indazol-
4-yl}phenyl)-N′-(3-methylphenyl)urea (34e). Compound 34e was
1
1
compound (122 mg, 97%). H NMR (DMSO-d6) δ 3.30 (s, 3H),
prepared from 30 using the same procedures as for 34a. H NMR
3.64-3.69 (m, 2H), 4.20-4.28 (m, 2H), 7.37 (t, J ) 8.82 Hz, 1H),
7.77 (dd, J ) 8.98, 1.53 Hz, 1H).
(DMSO-d6) δ 1.93 (m, 2H), 2.24 (t, J ) 8.14 Hz, 2H), 2.29 (s,
3H), 3.55 (m, 2H), 3.62 (m, 2H), 4.27 (t, J ) 5.43 Hz, 2H), 6.73
(d, J ) 7.80 Hz, 1H), 6.80 (d, J ) 7.12 Hz, 1H), 6.86 (d, J ) 7.80
Hz, 1H), 7.16 (t, J ) 7.80 Hz, 1H), 7.25 (m, 1H), 7.32 (s, 1H),
7.35 (d, J ) 8.48 Hz, 2H), 7.56 (d, J ) 8.48 Hz, 2H), 8.64 (s, 1H)
8.78 (s, 1H); MS (ESI) m/z 485 (M + H)+.
1-{4-[3-Amino-7-(2-methoxy-ethoxy)-1H-indazol-4-yl]-phen-
yl}-3-m-tolyl-urea (34f). Compound 34f was prepared from 31f
using the same procedures as for 34a. 1H NMR (DMSO-d6) δ 2.29
(s, 3H), 3.35 (s, 3H), 3.76 (dd, J ) 5.09, 3.73 Hz, 2H), 4.27 (t, J
) 3.05 Hz, 2H), 4.29 (s, 2H), 6.67 (d, J ) 7.46 Hz, 1H), 6.79 (d,
J ) 7.80 Hz, 2H), 7.16 (t, J ) 7.63 Hz, 1H), 7.25 (m, 1H), 7.31 (s,
1H), 7.34 (d, J ) 8.48 Hz, 2H), 7.55 (d, J ) 8.48 Hz, 2H), 8.63 (s,
7-(2-Dimethylaminoethoxy)-4-iodo-1H-indazol-3-ylamine (32a).
A mixture of the 31a (834 mg, 2.5 mmol) and hydrazine
monohydrate (1.2 mL, 25 mmol) in 5 mL of n-butanol was heated
at 110 °C for 5 h and then concentrated. The residue was redissolved
in ethyl acetate, washed with water and brine, dried over MgSO4,
filtered, and purified by flash column chromatography on silica
gel eluting with 6% methanol in dichloromethane to provide the
1
title compound (468 mg, 54%). H NMR (DMSO-d6) δ 2.23 (s,
6H), 2.68 (t, J ) 5.76 Hz, 2H), 4.17 (t, J ) 5.76 Hz, 2H), 4.97 (s,
2H), 6.54 (d, J ) 7.80 Hz, 1H), 7.19 (d, J ) 7.80 Hz, 1H), 11.90
(s, 1H); MS (ESI) m/z 347 (M + H)+.

1596 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Dai et al.
1H), 8.76 (s, 1H), 11.83 (s, 1H); MS (ESI) MS m/z 432 (M + H)+.
Anal. (C24H25N5O3) C, H, N.
using the control and background readings for that plate. Inhibition
constants are the mean of two determinations performed with seven
concentrations of the test compounds.
3-(Bromomethyl)-2-fluoro-6-iodobenzonitrile (39). A mixture
of 26a (8.0 g, 30.6 mmol), N-bromosuccinimide (NBS; 6.54 g,
36.78 mmol), and benzoyl peroxide (0.5 g) in carbon tetrachloride
(100 mL) was heated to reflux for 36 h, during which additional
NBS (9.0 g, 50.6 mmol) and benzoyl peroxide (1.0 g) was added
in three portions. The suspension was filtered, and the filtrate was
concentrated. The residue was purified by flash column chroma-
tography on silica gel with 20% ethyl acetate in hexanes to provide
the title compound (4.83 g, 46%). 1H NMR (DMSO-d6) δ 4.71 (s,
2H), 7.66 (t, J ) 8.14 Hz, 1H), 7.92 (d, J ) 8.14 Hz, 1H).
2-Fluoro-6-iodo-3-(4-morpholinylmethyl)benzonitrile (40a). A
solution of 39 (710 mg, 2.09 mmol) and morpholine (0.546 mL,
6.25 mmol) in DMF (8 mL) was stirred at room temperature
overnight, poured into water, and extracted twice with ethyl acetate.
The combined extracts were washed with water and brine, dried
(MgSO4), filtered, and concentrated to provide the title compound
Enzyme-Linked Imminosorbent Assay (ELISA) of KDR
Cellular Phosphorylation. NIH3T3 cells stably transfected with
full length human KDR (VEGFR2) were maintained in a Dulbec-
co’s modified Eagle’s medium (DMEM) with 10% fetal bovine
serum and 500 µg/mL geneticin. KDR cells were plated at 20 000
cells/well into duplicate 96-well tissue culture plates and cultured
overnight in an incubator at 37 °C with 5% CO2 and 80% humidity.
The growth medium was replaced with serum-free growth medium
for 2 h prior to compound addition. Compounds in DMSO were
diluted in serum-free growth medium (final DMSO concentration
1%) and added to cells for 20 min prior to stimulation for 10 min
with VEGF (50 ng/mL). Cells were lysed by addition of RIPA
buffer (50 mM Tris-HCl (pH 7.4), 1% IGEPAL, 150 mM NaCl, 1
mM EDTA, and 0.25% sodium deoxycholate) containing protease
inhibitors (Sigma cocktail), NaF (1 mM), and Na3VO4 (1 mM),
and placed on a microtiter plate shaker for 10 min. The lysates
from duplicate wells were combined and 170 µL of the combined
lysate was added to the KDR ELISA plate. The KDR ELISA plate
was prepared by adding anti-VEGFR2 antibody (1 µg/well, R&D
Systems) to an unblocked plate and incubated overnight at 4 °C.
The plate was then blocked for at least 1 h with 200 µL/well of
5% dry milk in phosphate buffered saline (PBS). The plate was
washed two times with PBS containing 0.1% Tween 20 (PBST)
before addition of the cell lysates. Cell lysates were incubated in
the KDR ELISA plate with constant shaking on a microtiter plate
shaker for 2 h at room temperature. The cell lysate was then
removed and the plate was washed five times with PBST. Detection
of phospho-KDR was performed using a 1:2000 dilution of
biotinylated 4G10 antiphosphotyrosine (UBI, Lake Placid, NY),
incubated with constant shaking for 1.5 h at room temperature and
washed five times with PBST, and for detection, a 1:2000 dilution
of streptavidin-HRP (UBI, Lake Placid, NY) was added and
incubated with constant shaking for 1 h at room temperature. The
wells were then washed five times with PBST and K-Blue HRP
ELISA substrate (Neogen) was added to each well. Development
time was monitored at 650 nm in a SprectrMax Plus plate reader
until 0.4-0.5 absorbance units were obtained (approximately 10
min) in the VEGF only wells. Phosphoric acid (1 M) was added to
stop the reaction, and the plate was read at 450 nm. Percent
inhibition was calculated using the VEGF only wells as 100%
controls and wells containing 5 µM pan-kinase inhibitor as 0%
controls (no VEGF wells were used to monitor endogenous
phosphorylation state of the cells). IC50 values were calculated by
nonlinear regression analysis of the concentration response curve.
Each IC50 determination was performed with five concentrations
and each assay point was determined in duplicate.
1
(710 mg, 98%). H NMR (DMSO-d6) δ 2.33-2.41 (m, 4H), 3.52
(s, 2H), 3.53-3.59 (m, 4H), 7.52 (t, J ) 7.80 Hz, 1H), 7.87 (d, J
) 8.14 Hz, 1H); MS (ESI) m/z 347 (M + H)+.
4-Iodo-7-(4-morpholinylmethyl)-1H-indazol-3-amine (41a).
Compound 41a was prepared from 40a using the same procedure
as for 32a. 1H NMR (DMSO-d6) δ 2.34-2.42 (m, 4H), 3.54-3.60
(m, 4H), 3.58 (s, 2H), 6.85 (d, J ) 7.46 Hz, 1H), 7.30 (d, J ) 7.12
Hz, 1H), 11.66 (s, 1H); MS (ESI) m/z 359 (M + H)+.
4-(4-Aminophenyl)-7-(4-morpholinylmethyl)-1H-indazol-3-
amine (42a). Compound 42a was prepared from 41a and 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (10) using the same
1
procedure as for 33a. H NMR (DMSO-d6) δ 2.42 (d, J ) 4.07
Hz, 4H), 3.58 (m, 4H), 3.65 (s, 2H), 4.36 (s, 2H), 5.24 (s, 2H),
6.66 (dd, J ) 7.80, 4.41 Hz, 3H), 7.12 (m, 3H), 11.45 (s, 1H); MS
(ESI) m/z 324 (M + H)+.
N-{4-[3-Amino-7-(4-morpholinylmethyl)-1H-indazol-4-yl]phe-
nyl}-N′-(3-methylphenyl)urea (43a). Compound 43a was prepared
from 42a and m-tolyl isocyanate using the same procedure as for
22a. 1H NMR (DMSO-d6) δ 2.29 (s, 3H), 3.20-4.20 (m, 8H), 4.56
(s, 2H), 6.80 (d, J ) 7.12 Hz, 1H), 6.92 (d, J ) 7.12 Hz, 1H), 7.17
(t, J ) 7.80 Hz, 2H), 7.27 (d, J ) 7.23 Hz, 1H), 7.32 (s, 1H), 7.42
(m, 3H), 7.63 (d, J ) 8.48 Hz, 2H), 8.79 (s, 1H), 8.98 (s, 1H); MS
(ESI) m/z 457 (M + H)+.
N-(4-{3-Amino-7-[(4-methyl-1-piperazinyl)methyl]-1H-inda-
zol-4-yl}phenyl)-N′-(3-methylphenyl)urea (43b). Compound 43b
1
was prepared from 39 using the same procedures as for 43a. H
NMR (DMSO-d6) δ 2.29 (s, 3H), 2.79 (s, 3H), 3.00-3.50 (m, 8H),
3.95 (s, 2H), 6.80 (d, J ) 7.46 Hz, 1H), 6.84 (d, J ) 7.46 Hz, 1H),
7.17 (t, J ) 7.63 Hz, 1H), 7.23-7.29 (m, 2H), 7.32 (s, 1H), 7.39
(d, J ) 8.48 Hz, 2H), 7.61 (d, J ) 8.81 Hz, 2H), 8.76 (s, 1H), 8.94
(s, 1H); MS (ESI) m/z 470 (M + H)+. Anal. (C27H31N7O‚3.6CF3-
CO2H) C, H, N.
Estradiol-Induced Murine UE Assay. Twelve week old balb/c
female mice (Taconic, Germantown, NY) were pretreated with 10
units of Pregnant Mare’s Serum Gonadotropin (PMSG; Calbiochem)
intraperitoneally (ip) administered 72 and 24 h prior to estradiol.
Mice were randomized the day of the experiment. Test compounds
were formulated in a variety of vehicles and administered po 30
min prior to stimulation with an ip injection of water soluble 17â-
estradiol (20-25 µg/mouse). Animals were sacrificed and uteri
removed 2.5 h following estradiol stimulation by cutting just
proximal to the cervix and at the fallopian tubes. After the removal
of fat and connective tissue, uteri were weighed, squeezed between
filter paper to remove fluid and weighed again. The difference
between wet and blotted weights represented the fluid content of
the uterus. Compound-treated groups were compared to vehicle-
treated groups after subtracting the background water content of
unstimulated uteri. Experimental group size was five or six.
HT1080 Tumor Growth Inhibition Model. The 1080 human
fibrosarcoma cells were obtained from the American Type Tissue
Culture Collection and maintained in Dulbecco’s Modified Eagle
Medium supplemented with 10% fetal bovine serum and antibiotics.
For tumor xenograft studies, cells were suspended in PBS, mixed
with an equal volume of matrigel (phenol red free) to a final
Homogeneous Time-Resolved Fluorescence (HTRF) Assays
of Receptor Tyrosine Kinases (KDR, CSF1R, cKIT, FLT1, and
FLT3). Assays were performed in a total of 40 µL in 96-well Costar
black half-volume plates using HTRF technology.26 Peptide sub-
strate (Biotin-Ahx-AEEEYFFLFA-amide) at 4 µM, 1 mM ATP,
enzyme, and inhibitors was incubated for 1 h at ambient temperature
in 50 mM Hepes/NaOH pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 2.5
mM DTT, 0.1 mM orthovanadate, and 0.01% bovine serum
albumin. Inhibitors were added to the wells at a final concentration
of 3.2 nM to 50 µM with 5% DMSO added as cosolvent. The
reactions were stopped with 10 µL/well 0.5 M ethylenediamine-
tetraacetic acid (EDTA) and then 75 µL buffer containing strepta-
vidin-allophycocyanin (prozyme; 1.1 µg/mL) and PT66 antibody
europium cryptate (Cis-Bio; 0.1 µg/mL) was added to each well.
The plates were read from 1 to 4 h after addition of the detection
reagents and the time-resolved fluorescence (665 to 615 ratio)
measured using a Packard Discovery instrument. The amount of
each tyrosine kinase added to the wells was calibrated to give a
control (no inhibitor) to background (prequenched with EDTA) ratio
of 10-15 and was shown to be in the low nanomolar concentration
range for each kinase. The inhibition of each well was calculated

NoVel Tyrosine Kinase ActiVity Inhibitor ABT-869
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1597
auch, K.-H.; Schneider, M. R.; Drevs, J.; Martiny-Baron, G.; Totzke,
F.; Marme, D. PTK787/ZK222584, A Novel and Potent Inhibitor of
Vascular Endothelial Growth Factor Receptor Tyrosine Kinases,
Impairs Vascular Endothelial Growth Factor-induced Responses and
Tumor Growth after Oral Administration. Cancer Res. 2000, 60,
2178-2189.
concentration of 2 million cells/mL, and inoculated (0.25 mL) into
the flank of SCID-beige mice. One week after inoculation, tumor-
bearing animals were divided into groups (n ) 10), and administra-
tion of vehicle (2% EtOH, 5% Tween 80, 20% PEG 400, 73%
saline) or inhibitor at the indicted dose was initiated. Tumor growth
was assessed every 2-3 days by measuring tumor size and
calculating tumor volume using the formula [length × width2]/2.
Mouse PK Analysis. Male CD-1 mice weighing 26-30 g
(Charles River Labs) were dosed intraveneously via the tail vein
or orally by gavage with a metal feeder tube. Dosing solutions were
prepared in 2.5% ethanol, 2.5% DMSO, 5% Tween-80, 25%
PEG400, and pH 7.4 PBS, for a dosing volume of 10 mL/kg. Blood
samples were collected with a heparinized syringe by cardiac
puncture following CO2 asphixiation at specified times. Plasma
samples were aliquoted into 96-well plates, and proteins were
precipitated using acidified methanol. Supernatants were stored at
-20 °C. Sample analyses were performed by LC-MS using a
Shimadzu 10A-VP chromatography system with a Waters YMC-
AQ 5 cm column. The mobile phase consisted of 45% acetonitrile
and 0.1% acetic acid in water, and the flow rate was 0.4 mL/min.
Mass detection was accomplished with an ESI equipped LCQ-Duo
by ThermoFinnegan. External standards were prepared from spiked
control plasma and used to generate a response factor for every
study. Limits of detection were between 20 and 50 nM.
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and Microaneurysm Formation in PDGF-B-deficient Mice. Science
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Stimulating Factor 1 Promotes Progression of Mammary Tumors to
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(18) Adams, J.; Huang, P.; Patrick, D. A Strategy for the Design of
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(19) Arteaga, C. L. Molecular Therapeutics: Is One Promiscuous Drug
against Multiple Targets Better than Combinations of Molecule-
Specific Drugs? Clin. Cancer Res. 2003, 9, 1231-1232.
(20) Mandel, D. B.; Laird, A. D.; Xin, X.; Louie, S. G.; Christensen, J.
G.; Li, G.; Schreck, R. E.; Abrams, T. J.; Ngai, T. J.; Lee, L. B.;
Murray, L. J.; Carver, J.; Chan, E.; Moss, K. G.; Naznedar, J. O.;
Sukbuntherng, J.; Blake, R. A.; Sun, L.; Tang, C.; Miller, T.;
Shirazian, S.; McMahon, G.; Cherrington, J. M. In Vivo Antitumor
Activity of SU11248, A Novel Tyrosine Kinase Inhibitor Targeting
Vascular Endothelial Growth Factor and Platelet-derived Growth
Factor Receptors: Determination of a Phamarcokinetic/Pharmaco-
dynamic Relationship. Clin. Cancer Res. 2003, 9, 327-337.
(21) Wilhelm, S. M.; Carter, C.; Tang, L.; Wilkie, D.; McNabola, A.;
Rong, H.; Chen, C.; Zhang, X.; Vincent, P.; McHugh, M.; Cao, Y.;
Shujath, J.; Gawlak, S.; Eveleigh, D.; Rowley, B.; Liu, L.; Adnane,
L.; Lynch, M.; Auclair, D.; Taylor, I.; Gedrich, R.; Voznesensky,
A.; Riedl, B.; Post, L. E.; Bollag, G.; Trail, P. A. BAY 43-9006.
Exhibits Broad Spectrum Oral Antitumor Activity and Targets the
RAF/MEK/ERK Pathway and Receptor Tyrosine Kinases Involved
in Tumor Progression and Angiogenesis. Cancer Res. 2004, 64,
7099-7109.
(22) Dai, Y.; Guo, Y.; Frey, R. R.; Ji, Z.; Curtin, M. L.; Ahmed, A. A.;
Albert, D. H.; Arnold, L.; Arries, S. S.; Barlozzari, T.; Bauch, J. L.;
Bouska, J. J.; Bousquet, P. F.; Cunha, G. A.; Glaser, K. B.; Guo, J.;
Li, J.; Marcotte, P. A.; Marsh, K. C.; Mosley, M. D.; Pease, L. J.;
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Acknowledgment. We thank the Department of Structural
Chemistry, Abbott Laboratories, for recording the NMR and
MS spectral data.
Supporting Information Available: Spectral data of urea
boronates 16b-s and synthesis and spectral data of 44-47. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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